Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a recombinant microorganism having an enhanced ability to produce ethanol and butanol and a method for preparing ethanol and butanol using the same, and more particularly to a recombinant microorganism having an enhanced ability to produce ethanol and butanol, into which a gene encoding CoA transferase and a gene encoding alcohol/aldehyde dehydrogenase are introduced, and a method for preparing ethanol and butanol using the same.
• ethanol(C 2 H 5 OH) has been prepared by a method of fermenting starch or sugars, and most alcoholic beverages theses days are prepared by such a method.
• ethanol is currently being prepared by synthetic methods comprising using ethylene (ethene) obtained from petroleum as a raw material: a sulfuric acid hydrolysis method in which ethylene is absorbed into sulfuric acid to produce the sulfuric acid ester of ethanol, then hydrolyzed to produce ethanol together with diethyl ether, and a direct hydration method in which ethylene in a gaseous phase is allowed to react with aqueous vapor by contact using a solid phosphoric acid catalyst, thereby leading to direct synthesis of ethanol.
• said methods have disadvantages i in that petroleum is a basic raw material, and that in the case of the sulfuric acid hydrolysis method, large scale facilities are required for the concentration and circulation of a large amount of sulfuric acid.
• butanol C 4 H 9 OH
• ethanol chemical synthesis of butanol also uses petroleum as a raw material to produce propylene, which is used to synthesize butanol by the oxo process.
• propylene Such a method involving high temperature and high pressure, using petroleum as a raw material, is inefficient in both cost and energy (Tsuchida et al, Ind. Eng. Chem. Res., 45:8634, 2006).
• a plasmid (pFNK6) was prepared by introducing 3 genes: a gene ⁇ adc) encoding acetoacetic acid decarboxylase, a gene (ctfA) encoding CoA transferase A and a gene (ct ⁇ B) encoding CoA transferase B into a vector and constructing an artificial operon using an adc promoter, and the plasmid was introduced into Clostridium acetobutylicum ATCC 824, thereby improving the productivity of acetone, butanol and ethanol by 95%, 37% and 90%, respectively, compared to the wild-type (Mermelstein et al, Biotechnol.
• Clostridium beijerinckii BAlOl which is a mutant strain obtained through random mutagenesis, was fermented using maltodextrins as a carbon source, and was reported to have produced 18.6 g/1 of butanol (Ezeji et al, Appl Microbiol. Biotechnol, 63:653,
• the present inventors have made extensive efforts to develop microorganisms capable of producing ethanol and butanol with high yield without producing byproducts based on the pathway for ethanol and butanol synthesis (FIG. 1), and as a result, constructed a recombinant microorganism by cloning two enzymes derived from Clostridium acetobutylicum ATCC 824: (1) ctfAS> encoding CoA transferase, which converts acetic acid and butyric acid into acetyl CoA and butylyl CoA, respectively, and (2) adhEl encoding alcohol/aldehyde dehydrogenase, which converts acetyl CoA and butyryl CoA into ethanol and butanol, respectively, and introducing the cloned genes into a host microorganism incapable of producing organic solvents, and confirmed that the recombinant microorganism produces high concentrations of ethanol and butanol while producing almost no acetone as
• Another object of the present invention is to provide a method for preparing ethanol and butanol using said recombinant microorganism.
• the present invention provides a method for constructing a recombinant microorganism having an enhanced ability to produce ethanol and butanol, the method comprises introducing a gene encoding an enzyme that converts acetic acid and butyric acid to acetyl CoA and butylyl CoA, respectively; and/or a gene encoding an enzyme that converts acetyl CoA and butyryl CoA to ethanol and butanol, respectively, into a host microorganism which has genes encoding enzymes involved in the biosynthetic pathway for conversion of acetyl CoA to butyryl CoA.
• the present invention also provides a recombinant microorganism having an enhanced ability to produce ethanol and butanol, which has a gene encoding an enzyme that converts acetic acid and butyric acid to acetyl CoA and butylyl CoA, respectively; and/or a gene encoding an enzyme that converts acetyl CoA and butyryl CoA to ethanol and butanol, respectively, introduced or amplified into a host microorganism having genes encoding enzymes involved in the biosynthetic pathway for conversion of acetyl CoA to butyryl CoA.
• the present invention provides a method for preparing ethanol and/or butanol, the method comprising the steps of culturing said recombinant microorganism and recovering ethanol and/or butanol from the culture broth.
• FIG. 1 is a schematic diagram showing the metabolic pathway in a degenerated strain of Clostridium acetobutylicum (A), which has no ability to produce ethanol and butanol, and the metabolic pathway for the synthesis of ethanol and butanol in a recombinant strain constructed by introducing ctf AB and adhEl into the degenerated strain (B).
• FIG. 2 is a genetic map of the recombinant vector pIMPl ::adhEl .ctfAB which contains ctf AB and adhEl.
• the present invention relates to method for constructing a recombinant microorganism having an enhanced ability to produce ethanol and butanol, the method comprises introducing or amplifying a gene encoding an enzyme that converts acetic acid and butyric acid to acetyl CoA and butylyl CoA, respectively; and/or a gene encoding an enzyme that converts acetyl CoA and butyryl CoA to ethanol and butanol, respectively, into a host microorganism which has genes encoding enzymes involved in the biosynthetic pathway for conversion of acetyl CoA to butyryl CoA.
• the present invention also relates to a recombinant microorganism having an enhanced ability to produce ethanol and butanol, which has a gene encoding an enzyme that converts acetic acid and butyric acid to acetyl CoA and butylyl CoA, respectively; and/or a gene encoding an enzyme that converts acetyl CoA and butyryl CoA to ethanol and butanol, respectively, introduced or amplified into a host microorganism having genes encoding enzymes involved in the biosynthetic pathway for conversion of acetyl CoA to butyryl CoA.
• the term "amplification” is used herein broadly to refer to processes: mutation, substitution or deletion, and insertion of some base(s) of a relevant gene; or introducing a gene derived from other microorganism encoding the same enzyme to increase the activity of the corresponding enzyme.
• said biosynthetic pathway for conversion of acetyl CoA into butyryl CoA is preferably [acetyl CoA ⁇ acetoacetyl CoA ⁇ 3- hydroxybutyryl CoA ⁇ crotonyl CoA ⁇ butyryl CoA].
• the host microorganism preferably has an acetone biosynthetic pathway blocked and thus has acetone production of less than 10% of the total organic solvent production.
• An adc (a gene encoding acetoacetic acid decarboxylase) may be deleted in said pathway for acetone biosynthesis, but is not limited thereto.
• said host microorganism is preferably derived from the genus Clostridium, but it is not limited thereto as long as it has a biosynthetic pathway for conversion of acetyl CoA into butyryl CoA.
• the enzyme converting acetic acid and butyric acid into acetyl CoA and butylyl CoA, respectively, is CoA transferase; and the gene encoding the CoA transferase is ctf. AB.
• the enzyme converting acetyl CoA and butyryl CoA into ethanol and butanol, respectively is alcohol/aldehyde dehydrogenase; and the gene encoding the alcohol/aldehyde dehydrogenase is adhEl.
• the present invention used only said ctf AB and adhEl derived from Clostridium acetobutylicum ATCC 824 as an example, but genes derived from other microorganisms may be used without limitation as long as they are expressed in a host cell, into which they are introduced, and have the same activities.
• the host microorganism used is a mutant M5 strain of Clostridium acetobutylicum which lacks megaplasmid (carrying 127 genes, including a gene encoding acetoacetic acid decarboxylase, a gene encoding CoA transferase and a gene encoding alcohol/aldehyde dehydrogenase).
• the mutant M5 strain of Clostridium acetobutylicum is a microorganism whose pathway for acetone biosynthesis is blocked (FIG. 1).
• Clostridium acetobutylicum M5 was used as an example of the host microorganisms of the genus Clostridium whose pathway for acetone biosynthesis is blocked, but Clostridium acetobutylicum INYG, 4NYG, 5NYG and DGl (Stim- Herndon, K.P. et al, Biotechnol. /Food Microbiol., 2: 11, 1996), C. acetobutylicum ATCC 824 Type IV, M3, M5, 2-BB R, 2-BB D, Rif B 12, Rif DlO, Rif F7, and C. butyricum ATCC 860 (Clark, S. W. et al, Appl. Environ.
• Microbiol, 55:970, 1989 may also be used.
• the recombinant microorganism M5 pIMPl ::adhEl .ctfAB
• pIMPl ::adhEl .ctfAB a recombinant vector carrying said ctf AB and adhEl
• the present invention relates to a method for preparing ethanol and/or butanol, the method comprising the steps of culturing said recombinant microorganism and recovering ethanol and/or butanol from the culture broth.
• the processes of culturing recombinant microorganisms and recovering ethanol and butanol may be performed using the conventional o culture method and the conventional method for isolation and purification of ethanol/butanol known in the fermentation art.
• the recovery of butanol and ethanol is usually carried out after completing the culture, it may be carried out during culture in order to improve productivity, using proper methods such as gas-stripping method (Thaddeus et al., Bioprocess Biosyst. Eng., 27:207, 2005). That is, continuous culture while recovering ethanol and butanol produced during the culture is also within the scope of the present invention.
• the following examples illustrate a specific mutant strain of Clostridium acetobutylicum M5 as a host strain incapable of producing organic solvents, but it will be apparent to one skilled in the art that other microorganisms of the genus Clostridium or of other genera, which have biosynthetic pathways for conversion of acetyl CoA to butyryl CoA and whose pathways for organic solvent biosynthesis are blocked can be used as a host strain, and the same genes can be introduced into the host strain for ethanol and butanol production.
• Example 1 Preparation of a recombinant vector containing adhEl gene encoding alcohol/aldehyde dehydrogenase, and ctfAB gene encoding CoA transferase
• the adhEl, ctfA and ctfB genes of Clostridium acetobutylicum ATCC 824 which have the base sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, were cloned together with the promoter and transcription termination sequences thereof.
• PCR (Table 1) was performed with the primers of SEQ ID NO: 1 and SEQ ID NO: 2, then the obtained adhEl, ctfA and ctfB genes were cut with the restriction enzyme Sail and inserted into Clostridium/E. coli shuttle vector pIMPl (Mermelstein, L.D.
• the base sequences of the cloned adhEl and ctfAB genes derived from Clostridium acetobutylicum ATCC 824, were analyzed, and the amino acid sequences of alcohol/aldehyde dehydrogenase and CoA transferase were deduced. As the result, the DNA sequences (SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5) and amino acid sequences (SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8) of the adhEl and ctfAB of Clostridium acetobutylicum ATCC 824 were identified.
• M5(pIMPl ::adhEl . ctfAB) strain was constructed by introducing the recombinant vector pIMPl ::adhEl .ctfAB constructed in Example 1 into Clostridium acetobutylicum M5 strain by electroporation.
• the recombinant vector of Example 1 was introduced into Escherichia coli TOPlO, which contains the vector p ANl expressing Bacillus subtilis Phage ⁇ 3T I methy transferase (Mermelstein et al, Appl. Environ. Microbiol, 59: 1077, 1993) to induce methylation thereof, such that the vector becomes suitable for transformation into Clostridium.
• the methylated vector was isolated and purified from E. coli, and then introduced into a mutant strain of Clostridium acetobutylicum M5 (Cornillot et al, J. Bacteriol, 179:5442, 1997) which lacks megaplasmid (carrying 176 genes, including a gene encoding acetoacetic acid decarboxylase, a gene encoding CoA transferase and a gene encoding alcohol/aldehyde dehydrogenase), thus preparing a recombinant microorganism.
• pIMPl which had been used as a cloning vector, was introduced into Clostridium acetobutylicum M5 strain, thus preparing M5(pIMPl) strain.
• M5 competent cells were prepared for transformation as follows: First, M5 strain was inoculated into 10m£ of CGM (Table 2) and cultured to an OD of 0.6. The culture broth was inoculated into 60m# of 2X YTG medium (Bacto tryptone 16g, Yeast extract 1Og, NaCl 4g and Glucose 5g per 1 liter) to a concentration of 10% and the cells were cultured for 4-5 hours. The microorganism cells were washed twice with transformation buffer (EPB, 27OmM sucrose 15m£, 686mM NaH 2 PO 4 HO ⁇ , pH 7.4) and then suspended in 2.4ml, of the same buffer.
• transformation buffer EPB, 27OmM sucrose 15m£, 686mM NaH 2 PO 4 HO ⁇ , pH 7.4
• the thus prepared 600 ⁇ i of the M5 competent cells were mixed with 25 /z£ of the recombinant plasmid DNA, and the mixture was loaded into a cuvette with a 4mm electrode gap, and then was subjected to electric shock at 2.5 kV and 25uF, followed by suspending immediately in ImI of 2X YTG medium to culture for 3 hours at 37 ° C ; thus, selecting transformants by spreading on a solid 2X YTG medium containing 40 ⁇ g/mi of erythromycin.
• Example 3 Production of ethanol/butanol using the recombinant microorganism M5(pIMPl : : adhE 1.ctf AB)
• Example 2 The recombinant microorganism M5(pIMPl ::adhEl.ctfAB) prepared in Example 2 was cultured to examine the performance.
• CGM medium was sterilized, taken out at a temperature higher than 80 ° C , charged with nitrogen gas, and cooled to room temperature in an anaerobic chamber. Then,
• the glucose in the medium was measured using a glucose analyzer (model2700 STAT, Yellow Springs Instrument, Yellow Springs, Ohio, USA); and an aliquot of the medium was taken out at various time points in order to measure the concentrations of acetone, ethanol and butanol produced therefrom, using a gas chromatography (Agillent 6890N GC System, Agilent Technologies Inc., CA, USA) equipped with a packed column(Supelco CarbopackTM B AW/6.6% PEG 2OM, 2 m x 2 mm ID, Bellefonte, PA, USA).
• the present invention has the effect of providing a recombinant microorganism having the ability to produce ethanol and butanol with high yield through the introduction or amplification of specific genes.
• the recombinant microorganism according to the present invention shows not only almost no production of byproducts such as acetone, but also enhanced ethanol and butanol productivity per unit hour. Accordingly, the inventive microorganism is useful for industrial production of ethanol/butanol.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=40801685

Family Applications (1)

Country Status (7)

Families Citing this family (13)

Family Cites Families (5)

• 2007
  • 2007-12-20 KR KR1020070134701A patent/KR101076042B1/ko active Active
• 2008
  • 2008-12-22 AU AU2008341277A patent/AU2008341277A1/en not_active Abandoned
  • 2008-12-22 US US12/809,323 patent/US20110027845A1/en not_active Abandoned
  • 2008-12-22 CN CN2008801263685A patent/CN101952430B/zh active Active
  • 2008-12-22 JP JP2010539309A patent/JP2011507504A/ja active Pending
  • 2008-12-22 WO PCT/KR2008/007577 patent/WO2009082148A2/en not_active Ceased
  • 2008-12-22 EP EP08865113A patent/EP2222845A4/de not_active Withdrawn

Also Published As

Similar Documents

Legal Events